pISSN 2466-1384 eISSN 2466-1392 大韓獸醫學會誌 (2016) 第 56 卷 第 2 號 Korean J Vet Res(2016) 56(2) : 129~131 http://dx.doi.org/10.14405/kjvr.2016.56.2.129 <Case Report> Salmonella enterica subsp. enterica infections in eastern great egrets (Ardea alba modesta) Hansol Jeong1, Geewook Shin1, Seungwon Yi1, Eunju Kim2, Haebeom Lee2,3, Myeon-Sik Yang1, Chae-Woong Lim1, Bumseok Kim1,* 1Veterinary Diagnostic Center, and 2Chonduk Wildlife Rescue & Conservation Center, College of Veterinary Medicine, BK21 Plus Program and Bio-Safety Research Institute, Chonbuk National University, Iksan 54596, Korea 3College of Veterinary Medicine, Chungnam National University, Daejeon 34134, Korea. (Received: February 11, 2016; Revised: April 14, 2016; Accepted: May 23, 2016) Abstract : Five eastern great egrets with a history of ataxia, wry neck, and wet feathers were submitted to the Veterinary Diagnostic Center for pathologic examination. Slightly enlarged livers with diffuse white-grayish nodules were observed. Microscopically, the hepatic and lung parenchyma contained granulomatous lesions consisting of central necrosis. Some hearts showed myofiber necrosis with infiltration of histiocytes and heterophils. Partial 16SrRNA and gyrB gene sequences of all isolates showed high similarities (99–100%) to those of Salmonella (S.) enterica subsp. enterica. Based on pathological and molecular biological results, S. enterica subsp. enterica systemic infections were diagnosed in eastern great egrets of Korea. Keywords : Korea, Salmonella, egret, infection Salmonella spp. is a group of gram-negative facultative egret presented with ataxia, wry neck, and wet feathers. The anaerobic pathogenic bacteria that has been isolated from a juvenile egret was euthanized due to poor prognosis and variety of mammals and avian species. Salmonellosis out- debilitated condition. Since 9 birds were severely decom- breaks have been reported in various wild birds including posed, only five birds (four dead birds and one euthanized gulls, ducks, and passerine birds [11]. Salmonellosis in wild bird) were submitted to the Veterinary Diagnostic Center of birds has long been recognized as a potential health risk to Chonbuk National University for pathologic examination. humans and livestock [6, 10]. Salmonella (S.) enterica subsp. At postmortem examination, no ectoparasites or skin enterica infections appear as primary enteropathogens that lesions were found. Grossly, the main lesions were slightly cause gastroenteritis and serious systemic infections in young enlarged livers with multifocal white-grayish nodules, and or emaciated birds [3]. there were no lesions in other organs (Fig. 1A). Swabs from The eastern great egret (Ardea alba modesta), a white blood, liver, and feces were cultured on blood agar plates heron in the genus Ardea, is a subspecies of the great egret (BAP) and tryptic soy agar (TSA) at 37oC for 24 h and sub- (Ardea alba). There have been several reports about egrets cultured to MacConkey agar. Single colonies from the liver infected by viruses, including eastern equine encephalitis and fecal cultures appeared white with no hemolysis on BAP virus [2], Newcastle disease virus [13], and influenza virus and MacConkey agar. Colonies were separately re-cultured [15]. A previous report about cattle egrets in Texas showed on TSA, and the isolates were stored at −70oC using Cryo- 17 S. enterica subsp. enterica serotypes that were isolated in care Bacteria Preservers (Key Scientific Products, USA). the digestive tracts, spleens, and livers of nestling egrets [7]. DNA was extracted from all isolates using a DNA mini kit A previously published study suggests that Salmonella infec- (Qiagen, Germany). After the isolated pathogen was pre- tion in herons is much more common than expected [5]. We sumptively identified by partial 16SrRNA sequencing, the report here the first cases of S. enterica subsp. enterica infec- primer sequences, PCR amplification, and gyrB gene sequenc- tions in eastern great egrets of Korea. ing were performed in accordance with a previous study [1]. In August 2012, 13 dead eastern great egrets (12 juveniles DNA fragments were sequenced and analyzed by BLAST and one adult) were found along with one live juvenile egret search and BLAST distance tree (National Center for Bio- in a Dukjin-pond in the urban area of Jeonju City. The live technology Institute, USA). *Corresponding author Tel: +82-63-850-0953, Fax: +82-63-850-0910 E-mail: [email protected] 129 130 Hansol Jeong, Geewook Shin, Seungwon Yi, Eunju Kim, Haebeom Lee, Myeon-Sik Yang, Chae-Woong Lim, Bumseok growth of the suspected pathogens. A BLAST search using partial 16SrRNA and gyrB gene sequences revealed that the etiologic bacteria were S. enterica subsp. enterica, as expected. Results showed 100% query coverage and high similarities (99–100%) with 16SrRNA (GenBank No. CP002614.1) and gyrB (GenBank No. CP006602.1) gene sequences of S. enter- ica subsp. enterica. AST results indicated that the present isolates were susceptible to all antimicrobial drugs tested. All submitted dead egrets showed systemic inflammation including necrotic foci and infiltration of heterophils and lymphocytes in the intestine, kidney, lung, and liver. Previ- ous reports have proven these as characteristics of salmonel- losis [5, 7, 8]. A previous report of Salmonella in cattle egrets showed a high incidence in young chicks with severe hepatitis characterized by necrotic cores with inflammatory cells [7]. This case also characteristically showed severe hep- atitis with diffuse necrosis surrounded by mononuclear cells. The gyrB gene encodes the subunit B protein of DNA gyrase (topoisomerase type II), which is an essential enzyme for DNA replication distributed universally among bacterial species. The gyrB gene sequences have been used widely in phylogenetic studies of Salmonella, Shigella, and Escheri- Fig. 1. Representative gross and microscopic lesions from chia coli [1]. Previous studies of gyrB show that the gyrB egrets infected by Salmonella. (A) Multifocal distribution of gene is a suitable marker for investigating phylogenetic and white-grayish nodules (arrowheads) was observed in livers. (B) taxonomic relationships at the species level. The rate of Necrotic foci surrounded by lymphocytes, liver. (C) Granulo- molecular evolution inferred from gyrB gene sequences is matous lesions with central necrosis in parenchyma, lung. (D) higher than that inferred from 16SrRNA gene sequences. Interstitial nephritis with infiltrations of mononuclear cells and Therefore, gyrB gene sequence analysis is considered a more heterophils, kidney. (E) Focal necrosis of villi and inflamma- accurate method for identifying unknown bacteria than tion, (insert: mononuclear cell infiltration in lamina propria) small intestine. (F) Myofiber necrosis with infiltration of histi- 16SrRNA sequences [1, 14]. Although gyrB has limitations in ocytes and heterophils, heart. H&E stain. Scale bars 50 µm (C identifying genes at a serotype level, it can reliably confirm and E), =20 µm (B, D and F). 1,000× (insert in E). genes to the subspecies level. Based on the use of gyrB to distinguish species, it is concluded that the gram-negative bacteria found in the eastern great egrets are S. enterica An antibiotic susceptibility test (AST) was performed by subsp. enterica. the disc diffusion method using 9 different antibiotics (ami- In the current case, a group of the same species died in a kacin, gentamicin, enrofloxacin, ampicillin, amoxicillin/cla- limited area at the same time. S. enterica subsp. enterica was vualnic acid, cefazolin, cefotazime, sulfamethoxazole/trimetho- the only infectious bacteria found in common in these ani- prime, and doxycycline) according to Clinical and Labora- mals. Moreover, previous studies have indicated that salmo- tory Standards Institute document M100-S17. Tissue sam- nellosis results in high mortality in certain species of birds [9, ples from the heart, lung, liver, kidney, spleen, and intestine 12]. Eastern great egrets might be vulnerable to Salmonella were collected, fixed in 10% neutral buffered formalin, and infection. processed routinely for histopathology. Tissues sectioned at 6 Because salmonellosis is a zoonotic disease, Salmonella µm were stained with hematoxylin and eosin (H&E) for his- infection in wild birds is a potential health risk to the public. topathological examination. Accumulating evidence has shown that S. enterica subsp. Microscopically, there were granulomatous lesions in the enterica infections in wild birds disseminate bacteria and hepatic and lung parenchyma consisting of central necrosis cause salmonellosis in livestock, dogs, cats, and humans [6, surrounded by lymphocytes (Fig. 1B and C). The kidneys 10, 11]. When Salmonella spreads to a farm or any place were hyperemic and had interstitial nephritis with infiltra- containing livestock, it can persist to cause lasting economic tions of mononuclear cells and heterophils (Fig. 1D). Focal losses. In the current case, dead egrets were found in a pond villi necrosis with mononuclear cell infiltration was also of an urban area, which could have caused infection in observed in the lamina propria of the small intestine (Fig. humans or pet animals. Despite the latent danger to humans, 1E). Two of the five hearts showed myofiber necrosis with there are not many cases or studies regarding salmonellosis infiltration of histiocytes and heterophils (Fig. 1F). in wild birds in Korea. Wild birds may function as a critical Bacterial cultures from blood and liver